Abstract
The ability to non-invasively detect specific damage to the kidney has been limited. Identification of extracellular vesicles released by cells, especially when under duress, might allow for monitoring and identification of specific cell types within the kidney that are stressed. We have adapted a previously published traditional flow cytometry method for use with an imaging flow cytometer (Amnis FlowSight) for identifying EV released by specific cell types and excreted into the urine or blood using markers characteristic of particular cells in the kidney. Here we present a protocol utilizing the Amnis FlowSight Imaging Flow Cytometer to identify and quantify EV from the urine of patients with essential hypertension and renovascular disease. Notably, EV isolated from cell culture media and plasma can also be analyzed similarly.
Keywords: Exosomes, Extracellular vesicles, Imaging flow cytometry, Biomarkers, Hypertension, Renovascular disease
Background
Extracellular vehicles (EVs) are released from cells under normal conditions and their numbers are known to increase when the cells are exposed to stress conditions. Therefore, levels of urinary EVs have been shown to be associated with various kidney disorders such as polycystic kidney disease (Hogan et al., 2009), acute kidney injury (Aghajani Nargesi et al., 2017; Cappuccilli et al., 2018), and various glomerular diseases (Zhang et al., 2019).We have previously shown that levels of EVs that are increased in patients with hypertension likely originate from podocytes (Kwon et al., 2017) and peritubular capillaries (PTC) (Sun et al., 2018; Zhang et al., 2019). We have also recently shown that urinary levels of EVs reflecting renal cellular senescence are altered in these patients (Santelli et al., 2019). We describe here a more detailed protocol that can be used for the isolation and quantitative characterization of EVs isolated from cell culture media, plasma, and urine (Kwon et al., 2017; Sun et al., 2018; Conley et al., 2019; Zhang et al., 2019). The primary advantages of this method of EV isolation are shorter processing time and obviating the requirement for an ultracentrifuge to isolate the EVs.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Recipes
Acknowledgments
This protocol was adapted from Sun et al. (2018). This work was partly supported by the National Institutes of Health grant numbers DK10081, DK104273, DK102325, and DK120292.
Competing interests
No competing interests.
Ethics
This study was approved by the institutional review board of the Mayo Clinic, and performed in accordance with the ethical principles of the Declaration of Helsinki. Informed written consent was obtained from each patient.
References
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